This proposal focuses on a novel enzymatic pathway that reduces short-chain aldehydes, eliminating toxic byproducts of oxidative stress in photoreceptor cells. Previous studies have shown that photoreceptor retinol dehydrogenases RDH11 and RDH12 are able to reduce short-chain aldehydes and hydroxyaldehydes of various lengths and unsaturation. These molecules are end products of the lipid peroxidation of polyunsaturated fatty acids. 4-hydroxy-trans-2-nonenal (4-HNE) is one of the major aldehydic products and is an inducer and mediator of oxidative stress. The specificity of RDH11 and RDH12 for the short-chain (hydroxy)aldehydes has been largely overlooked until now and recent findings have prompted us to re-evaluate its significance for the function of these enzymes in the retina. First, it was found that 4-HNE-protein adducts accumulate in photoreceptors during oxidative stress. Then, it was found that the Rdh12 knockout mice are more sensitive to light-induced oxidative damage. Finally, we found a protective effect of RDH11 and RDH12 against 4-HNE-induced apoptosis and adduct formation in cell culture. Based on these findings, we hypothesize that RDH11 and RDH12 are effective at reducing 4-HNE and are therefore an important detoxification system in photoreceptor cells. We will use the Rdh11 and Rdh12 knockout mice to investigate the following Specific Aims: Aim 1 is to characterize the catalytic activities of RDH11 and RDH12 towards 4-HNE in vitro and in mouse retina. We will characterize the kinetics of 4- HNE reduction using microsomal fractions of cells transfected with wild-type Rdh11 and Rdh12 or Rdh12 mutants as a source of enzyme and using a highly accurate and sensitive mass spectrometry method to quantify the substrate and product of the reaction. We will also use microsomal fractions prepared from wild type, Rdh11, and Rdh12 knockout retinas to measure the contribution of each enzyme to the reduction of 4-HNE in vivo. Aim 2 is to determine whether RDH11 and RDH12 are protective against the formation of 4-HNE-protein adducts and the apoptosis of photoreceptors ex vivo and in vivo. Rdh12 knockout mice were found to be more sensitive to light-induced apoptosis of photoreceptors than the wild-type mice, but the mechanism that induces higher sensitivity is unknown. To determine the roles of RHD11 and RDH12 in this process, we will induce oxidative stress in whole retinal cultures with H2O2 and 4-HNE as well as in mice with constant bright light exposure. We will then quantify 4-HNE-protein adducts and photoreceptor apoptosis in these retinas from wild-type, Rdh11, and Rdh12 knockout mice. PUBLIC HEALTH RELEVANCE: If we successfully demonstrate that the physiological role of RDH11 and RDH12 is to detoxify 4-HNE in photoreceptor inner segments, this finding will have a significant clinical impact. First, because it will provide a strategy to treat patients with the early onset retinal dystrophy Leber Congenital Amaurosis, caused by mutations of the RDH12 gene. Second, such detoxification pathways will represent a potential target to slow down the progression of a number of other retinopathies involving oxidative stress.